Tissue engineering is an emerging interdisciplinary which studies the tissues and organs of a biological organism or functional substitutes thereof using the principles and approaches of engineering science and bioscience. The basic principles and approaches of tissue engineering are that cells are absorbed in vivo or in vitro on a scaffold with good biocompatibility, which is prepared from synthetic polymers and/or natural polymers (e.g. extracellular matrix) and can be adhered by an organism gradually, to form a cell-biological materials complex. After the scaffold is degraded and absorbed, the complex performs corresponding functions at a specific anatomical position in the body, meanwhile, the host cells proliferate, secrete new extracellular matrix, and finally a new tissue or organ having a function and shape corresponding to those of the original anatomical position is formed, thereby achieving the purpose of repairing tissue shapes and rebuilding functions. Tissue engineering comprises three elements, viz., specific tissue cells, a scaffold and extracellular matrix. The scaffold not only plays a central role since it not only can provide a structural support for the specific cells, but also can function as a template to guide tissue regeneration and control tissue structure.
The extracellular matrix of tissues of animals including humans is a complex of nano-sized fibrous proteins, polysaccharides and proteoglycans, and may be imitated using nanofiber structures. With the advent of nano-era, reports on nanofiber increase rapidly. The preparation methods of nanofiber can be classified into chemical methods, physical methods and electrical methods. The chemical methods are based on the principle of molecular self-assembly by which small molecules with specific structures may be assembled into fibrous macromolecules. The physical methods are that the nanofiber can be obtained by performing lyophilization of L-polyactic acid solution to remove solvent utilizing special sol-gel property of L-polyactic acid when it reaches the liquid-liquid phase equilibrium through. It is difficult to obtain large quantities of products through the chemical method, and the physical methods are only limited to the preparation of L-polyactic acid nanofibers. The electrical methods, which utilize the electrospinning technique, are that a macromolecular solution is charged under a high-voltage electrostatic field and made into filaments during ejection toward a low-voltage electrical field. In theory, if there is a suitable solution system for polymers, they all can be made into nanofibers by using the electrospinning technique, and batch production can be preformed.
In 1934, Formhals (U.S. Pat. No. 1,975,504) firstly reported a patent for the electrospinning technique. However, only in the last decade, the application of electrospinning filaments in tissue repair has been studied. Therefore, the understanding of design and preparation of the electrospinning filaments and their in vivo or in vitro interaction with cells at molecular and cellular level is rather superficial. The successful application of the electrospinning filament in clinical practice has barely been reported.
Synthetic polymers used in the preparation of electrospinning filaments may be degradable aliphatic polyesters such as polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), and copolymers or mixture thereof. Under electrospinning conditions, these traditional materials can be conveniently made into polymer microfibers having a diameter from tens of nanometers to hundreds of nanometers, which are very similar to the major component of natural extracellular matrix, i.e. collagen, and have the following advantages: (1) imitating the structure of ECM in the body to the greatest extent; (2) having a higher porosity and extremely large volume-surface ratio, which facilitate the adhesion, differentiation, proliferation of cells, the secretion of ECM and the like; (3) perfectly controlling the thickness, three-dimensional structure and mechanical properties of electrospinning filaments by adjusting the concentration of solutions, electrospinning parameters and the like, and thereby facilitating cell growth, nutrient absorption and the excretion of metabolites; and (4) conveniently preparing electrospinning filaments from one or more the above-mentioned polymers.
Related studies have found that electrospinning of pure synthetic polymer has the following defects: (1) the filaments, compared with those prepared from natural polymers, lack cell recognition sites, and thus it is difficult for cells to adhere thereto; (2) the filaments inherently possess hydrophobicity and thus have poor hydrophilicity (for example, polycaprolactone (PCL) and polylactic acid-polycaprolactone (PLLA-CL) have a water contact angle of 109-120° and 109-133°, respectively), which seriously affects the adhesion of cells and subsequent cell activities; and (3) the degradation products of PLA and PGA are relatively strong acids (lactic acid or glycolic acid), and once these degradation products accumulates around a implant, delayed inflammatory reaction will appear in a few months or years. Thus, although electrospinning of synthetic polymer has a porosity which is 1-2 orders of magnitudes higher than conventional methods (such as air bubbling method and freeze drying method) and have a very high volume-surface ratio, the low hydrophilicity renders most pores empty, and thereby the three dimensional structure cannot be utilized efficiently.
To solve the above-mentioned problems, there is an urgent need to develop new electrospinning filaments having biological activities and functions. The biological activities refer to a material formed by the physical (such as mixing) or chemical (such as covalent immobilization) combination of biologically active substances and the structure or function of this material may, in vivo and/or in vitro, have positive effects on living cells, and facilitate the interaction between cells and the scaffold, such as proliferation, migration and maintenance of the shape and function of normal cells, which relates to the inherent biochemical properties of the used materials. Natural polymers such as proteins (collagen, silk fibroin, gelatin, elastin) and polysaccharides (chitosan, hyaluronic acid) are most ideal. In recent years, blood-derived fibrinogen (Fg) has drawn increasing attention. However, the major problems of natural polymers after electrospinning treatment lies in that: 1) they have low mechanical strength and are degraded with a too fast speed after implantation, and thus post-processing is often required, such as cross linking by glutaraldehyde vapor or UV irradiation, which finally renders the products swollen in water or inside the body without degradation; formaldehyde treatment, which drives a transition of fibres from random coil to beta sheet, improves the degree of crystallinity and decreases porosity, making them have a more compact structure; or alkali treatment on natural polysaccharide biopolymers (such as chitin and cellulose), which improves mechanical strength and prolongs degradation time; (2) after the above-mentioned post-processing, the mechanical strength of the natural polymers is improved significantly, but the largest problem brought about by such post-processing is that the degradation speed is reduced and the natural polymers even cannot be degraded. As a scaffold material, its major function is assisting in wound healing-related protein adsorption and cell adhesion thereto as a temporary transitional substance, and thereby the purpose of tissues remodeling may be achieved by cell ingrowth and the secretion of its own extracellular matrix. Thus, the reduction or loss of degradation speed will seriously affect subsequent tissue regeneration process.
The generation of composite electrospinning filaments brings a new idea for overcoming the defects of electrospinning of pure synthetic polymers and natural polymers and retaining their respective advantages. Composite electrospinning filaments may change the surface properties of scaffold materials easily and economically. In theory, they have the following advantages: in terms of physical aspect, they improve the hydrophility and mechanical strength of new composite scaffold materials; and in terms of a biological aspect, after biological molecules binding to the synthetic polymers, they may facilitate recognition of surfaces of the material by cells and facilitate or control many physiological activities of cells, such as adhesion, expansion, activation, migration, proliferation and differentiation.
Research results indicate that, although electrospinning filaments prepared from synthetic-natural polymers, compared with those prepared from pure natural polymers or natural polymers, have been improved in their physical or biological properties, they are still far away from clinical requirements. One of the reasons for this is that the contact of the electrospinning filaments prepared from synthetic polymers (such as polylactic acid-polycaprolactone (P(LLA-CL)), PLC or PCL) and natural biopolymers (such as collagen, elastin and chitosan), with an aqueous solution, often gives rise to shrinkage of composite scaffold materials with a shrinkage ratio of up to 20-50%. The change of this characteristic directly affects the porosity, degradation speed, wettability and the like of the electrospinning filaments. So far, there have been no reports on the successful clinical application of such biological composite scaffold materials.
CN101780292A discloses a Fg-based three-dimensional porous nano-scaffold and a method for preparing the same. The three-dimensional porous nano-scaffold is prepared from Fg and polylactic acid/polycaprolactone with a mass ratio (Fg:polylactic acid/polycaprolactone) of 1:5-12:5.
Fg, a biomacromolecule with a relative molecular weight of 340,000, is composed of three pairs of peptide chains (α-chain, β-chain, and γ-chain), and its subunits are linked together as a whole via three disulfide bonds. Since Fg is extracted from plasma, it has good histocompatibility. Meanwhile, Fg is degraded by fibrinolysin in a body, and degradation products are no longer involved in blood coagulation, and finally are eliminated by body tissues. The biological functions of Fg lie in that: (1) it has hemostatic effect, i.e. under physical conditions, Fg is converted to fibrin to form a blood clot, thereby achieving hemostatic effect; (2) it functions as a scaffold carrier of cells, i.e. a fibrin-based scaffold material may deliver cells to different defect or coloboma sites, for example, human smooth muscle cells may proliferate well inside or on the surface of a blood clot, and likewise, fibrin glue may make normal human-derived keratinocytes and fibroblasts have good proliferation results; (3) fibrin acts as a carrier for the delivery of cytokines and peptides in an active way: some growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor, may bind to fibrin with strong binding and meanwhile may slowly diffuse from a blood clot; and insulin-like growth factor 1 and transforming growth factor β may be directly embedded in fibrin scaffolds during polymerization, protecting those these growth factors from denaturation and degradation by proteasomes in vivo and in vitro. Fibrin, comprises RGDS and RGDF bioactive peptides at Aα572-575 and 95-98, respectively, with which cells interact via the mediation of integrin and induce cellular signal transduction; and in addition, fibrin may be linked with antibiotics, painkillers and the like, and when fibrinogen is used to stop bleeding and to seal tissues, local affection and pain may be controlled in 1-2 weeks, which happens to be the degradation period of fibrin clots and during which fibrin may retain many bioactive substances in an active and effective way and position them locally to facilitate tissue repairing.
Further studies indicate that electrospinning filaments prepared from P(LLA-CL) and Fg with the ratio of 10:0, 8:2 and 0:10 (P(LLA-CL): Fg) have the water contact angles of 110°, 95° and 65°, respectively (see, Chuanglong He, The potential applications of the preparation of Fg/polylactic acid-polycaprolactone hybrid nanofiber scaffolds in soft tissue engineering). Similar research results may also be found in Fabrication of fibrinogen/P(LLA-CL)hybrid nanofibrous scaffold for potential soft tissue engineering applications, Journal of Biomedical Materials Research A, 97A(3):339-347(2011). In modern material science, it is recognized that when a material has a water contact angle of more than 65°, the surface wettability of the material is hydrophobicity.
A lot of studies prove that, for both the degradation of a scaffold material and the regeneration of host tissues, the surface wettability of a solid material is an important factor for regulating the balance of the two processes. In the surface chemistry of a material, it is a common phenomenon that water can wet some surfaces, on the contrary, it cannot wet some other surfaces, but forms liquid drops thereon with a limited “contact angle”. Such solid surface wetting phenomenon has driven material scientists to conduct researches for almost three centuries. Generally, the surface wettability of a solid material is measured as water contact angles. The contact time between liquid drops and polymers greatly affects the measured value of a contact angle. The contact angle formed when the liquid drops contact the polymer surface for the very first time, is referred to as initial contact angle, which rapidly decreases within 10-20 minutes, and when the contact angle no longer changes over time and reaches a constant value, it is referred to as equilibrium contact angle. When the equilibrium contact angle between a solid surface and water is more than 65°, the solid surface is referred to as hydrophobic surface, and when the equilibrium contact angle is more than 150°, the solid surface is referred to as super-hydrophobic surface; and when the equilibrium contact angle is less than 55°, the solid surface is referred to as hydrophilic surface, and when the equilibrium contact angle is less than 5°, the solid surface is referred to as super-hydrophilic surface. It is believed that, after P(LLA-CL) and Fg are blended with a certain ratio, the cell recognition sites on the surface of the prepared electrospinning filaments are improved substantially, but the water contact angle decreases from 110° of pure P(LLA-CL) to 65°, and thus the obtained material still belongs to hydrophobic materials. Just as described above, the hydrophobicity of a material will hinder the degradation in vivo and vitro, protein absorption and cell adhesion, affect the ingrowth of cells, especially blood capillaries, and have the following impacts: oxygen, nutrients, antibodies, immune cells and related antibacterial substances cannot be supplemented effectively; acidic metabolites cannot be successfully eliminated timely; and microorganisms (such as skin or blood-derived bacteria) aggregate locally and cannot be inhibited and eliminated effectively, the occurrence rate of infection may thus be up to 20-30%. The decrease or loss of tissue regeneration speed and the occurrence of local infection are main reasons for the recurrence of tissue defect diseases after repairing (such as recurrence after a hernia repair and recurrence after a pelvic organ prolapse repair). Therefore, how to effectively improve the hydrophility of electrospinning filaments is a key problem to be solved.
In conclusion, although a patent has been disclosed for the principle of the electrospinning technique in 1934, the electrospinning biological composite scaffold material has drawn more and more attention since tissue engineering has been booming in the last decade. Theoretically, the material has a network structure similar to that of connective tissue of a body and thus should have a promising application prospect. However, so far, the successful application of electrospinning biological composite scaffold material in clinical practice has not been reported, the reasons for which is greatly related to the superficial understanding of structure materials of this kind. The problems to be solved mainly include (1) how to improve the protein and cell recognition site of synthetic polymers; (2) how to improve the surface property of a material, especially wettability; (3) how to effectively reduce the common shrinkage phenomenon of the electrospinning biological composite scaffold material after contacting with an aqueous solution; and (4) how to effectively prevent bacterial infections with a occurrence rate up to 20-30%, and a high recurrence rate.